1. Field of the Invention
This invention relates to a method for determining the concentration of an analyte in a sample and, in particular, to a method for determining the concentration electrochemically.
2. Description of the Related Art
It is particularly valuable to determine the concentration of a specific component in a sample which is of biological origin, for example blood. For this purpose a biosensor is used; use is made of a change in the oxidation state of a mediator which interacts with an enzyme which has reacted with the analyte to be determined. The oxidation state of the mediator is chosen so that it is solely in the state which will interact with the enzyme on addition of the substrate. The analyte reacts with a stoichiometric concentration of the mediator via enzyme. This causes the mediator to be oxidised or reduced (depending on the enzymatic reaction) and this change in the level of mediator can be measured by determining the current generated at a given potential.
Normally, a measurement is taken during the oxidation (or reduction) of the mediator by the enzyme as it reacts with the analyte. This can, though, give rise to unreliable results. It has been proposed, therefore, to wait for the reaction to go to completion and then to take a measurement. However, the value obtained changes with time such that it is generally necessary to take a number of readings and then to determine the concentration using either an algorithm or by integration of the area under the curve which corresponds to the plot of the values. This change in value is caused by the effect of diffusion which occurs essentially linearly from the electrode. Thus as some of the mediator is oxidised (or reduced) on the electrode more mediator diffuses to the electrode on a continuing basis. However, this linear diffusion results in depletion of electroactive material around the working electrode.
In order to determine the concentration it is necessary to first of all obtain a value in the absence of the analyte and to subtract this “background” value from the enhanced value which one obtains when the analyte is present. It will be appreciated that this procedure is both complicated and prone to error.
Recently, steps have been taken to reduce the size of biosensors by making use of a micro electrode. This can be defined as an electrode where at least one of the dimensions does not exceed 50 μm and is frequently 1 to 25 or 30 μm.
Micro electrodes were devised because they were perceived to be better for the measurement of very small currents. This is because the use of an array of micro electrodes gives a better signal to noise ratio than does a single electrode. Micro electrodes were therefore devised for direct current determination and they have not found utility as biosensors involving an enzyme and a mediator. It has, though, surprisingly been found that the use of a micro electrode can, if used in a particular way, overcome the disadvantages of macro electrodes.
That micro electrodes can be used for this purpose is particularly surprising for two principal reasons. First, the current enhancement due to the presence of the analyte over the background level for a micro electrode is of course very much smaller than for a macro electrode. Accordingly with decreasing size it is impossible to obtain any accurate measurements. Second, because the area of the electrode is very small in relation to the volume of the sample diffusion to the electrode surface no longer takes place linearly as with macro electrode but, rather, radially. The distance over which the reaction occurs in solution is large compared to the size of the microelectrodes such that one would expect there to be little or no catalytic enhancement. One would therefore expect with decreasing electrode size no meaningful measurement to be obtainable.
Under normal steady-state conditions, the average distance that the oxidised state of the mediator will diffuse before it reacts to reform the reduced state will be (DtL)1/2 where D is the diffusion coefficient of the oxidised state and tL is the reaction half life before the oxidised state is reformed. It will be appreciated that because the size of the micro electrode is small in comparison to this diffusion distance, there is normally very little current enhancement in the presence of the catalytic reaction. In other words, the presence of the analyte which promotes the catalytic reaction leads to only a small current increase above that initially present. The small magnitude of this “perturbation current” thus means that accurate analytical results cannot be obtained.